This invention relates to arrays, particularly polynucleotide arrays such as DNA arrays, which are useful in diagnostic, screening, gene expression analysis, and other applications.
Polynucleotide arrays (such as DNA or RNA arrays), are known and are used, for example, as diagnostic or screening tools. Such arrays include features (sometimes referenced as spots or regions) of usually different sequence polynucleotides arranged in a predetermined configuration on a substrate. The arrays, when exposed to a sample, will exhibit a binding pattern. The array can be interrogated by observing this binding pattern by, for example, by labeling all polynucleotide targets (for example, DNA) in the sample with a suitable label (such as a fluorescent compound), and accurately observing the fluorescent signal on the array. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the sample. Peptide arrays can be used in a similar manner.
Biopolymer arrays can be fabricated using either in situ synthesis methods or deposition of the previously obtained biopolymers. The in situ synthesis methods include those described in U.S. Pat. No. 5,449,754 for synthesizing peptide arrays, as well as WO 98/41531 and the references cited therein for synthesizing polynucleotides (specifically, DNA). Such in situ synthesis methods can be basically regarded as iterating the sequence of depositing droplets of: (a) a protected monomer onto predetermined locations on a substrate to link with either a suitably activated substrate surface (or with a previously deposited deprotected monomer); (b) deprotecting the deposited monomer so that it can now react with a subsequently deposited protected monomer; and (c) depositing another protected monomer for linking. Different monomers may be deposited at different regions on the substrate during any one iteration so that the different regions of the completed array will have different desired biopolymer sequences. One or more intermediate further steps may be required in each iteration, such as oxidation and washing steps.
The deposition methods basically involve depositing biopolymers at predetermined locations on a substrate which are suitably activated such that the biopolymers can link thereto. Biopolymers of different sequence may be deposited at different regions of the substrate to yield the completed array. Washing or other additional steps may also be used. Typical procedures known in the art for deposition of polynucleotides, particularly DNA such as whole oligomers or cDNA, are to load a small volume of DNA in solution in one or more drop dispensers such as the tip of a pin or in an open capillary and, touch the pin or capillary to the surface of the substrate. Such a procedure is described in U.S. Pat. No. 5,807,522. When the fluid touches the surface, some of the fluid is transferred. The pin or capillary must be washed prior to picking up the next type of DNA for spotting onto the array. This process is repeated for many different sequences and, eventually, the desired array is formed. Alternatively, the DNA can be loaded into a drop dispenser in the form of an inkjet head and fired onto the substrate. Such a technique has been described, for example, in PCT publications WO 95/25116 and WO 98/41531, and elsewhere. This method has the advantage of non-contact deposition.
In either method of fabrication, glass or other transparent material, is often used for the substrate. Such materials particularly lend themselves to linking of a nucleotide of a monomer or polymer. Further, in array fabrication, the quantities of DNA available for the array are usually very small and expensive. Sample quantities available for testing are usually also very small and it is therefore desirable to simultaneously test the same sample against a large number of different probes on an array. These conditions require use of arrays with large numbers of very small, closely spaced features. It is important in such arrays that features actually be present, that they are put down accurately in the desired pattern, are of the correct size, and that the DNA is uniformly coated within the feature. Normally, in an automated apparatus the features are deposited according to a target array pattern.
However, every component in an array deposition apparatus are subject to errors such as component failure or variances in its operating parameters within, or sometimes even outside of, normal tolerances for such component. For example, a dispensing head used to dispense fluid droplets to form the array, may have one or more jets which fail or which vary slightly in the size of the droplets dispensed, the orientation of the jets with respect to one another, or the orientation of the head itself in the apparatus may be slightly off from a nominal position. Whatever the error source, the result is that a target array pattern is not produced. That is, there is a discrepancy between the target array pattern and the actual array pattern deposited. These discrepancies can occur in each cycle of the in situ process, or during deposition of presynthesized polynucleotides.
The validity of the results of any test using an array, is dependant on knowing where the features are on the carrier substrate and if they were actually there on the substrate to begin with. A line scan camera can be used to observe droplets after their deposition during fabrication to reduce the possibility that during array use in a test, a reaction did not occur because a feature was missing or subject to some other error, thereby resulting in a false test result. However, observing the droplets during array fabrication can, as a practical matter, be difficult. For example, it is difficult to obtain sufficient reflected light from either the droplets or substrate surface to the camera sensor as the droplets move past the line scan camera. The amount of light the camera sensor is exposed to is inversely proportional to the speed of the objects being viewed. The faster the objects move, the less light reaches the camera, which can result in poor image contrast for reliable feature imaging. While the fabrication speed could be slowed to capture more light, this is undesirable from a manufacturing perspective and so the line scan camera should capture images at the running speed of the system. An additional issue with obtaining sufficient light to the camera is that when the substrate is glass or is otherwise transparent, and given that the droplets themselves may be transparent and colorless, most of the light will pass through the substrate and not make it back to the camera.
It would be useful then, to provide a means by which arrays can be fabricated by depositing droplets of monomer, polymer, or any other material used during array formation, and in which the deposition of the droplets can be accurately and rapidly observed even against a transparent substrate. It would also be useful if any such means is relatively simple to construct and offers little interference with other components of a deposition apparatus.
The present invention realizes that relatively good images can be obtained of deposited droplets of monomer, polymer (such as DNA, RNA, or peptides), or other fluids deposited on one side of a transparent substrate during array fabrication, by back lighting those droplets (that is, by providing illumination from the back side of the substrate). However, the present invention further realizes that back lighting during array fabrication is not particularly practical. For example, the substrate should preferably be firmly and precisely held in a known position at all times so that droplets will be deposited at least close to the expected locations, while still being supported on a back side. This implies some type of clamping and support structure across a back side of the substrate thereby making the to provision of any lighting system across the back side during array manufacture, difficult. Further, illuminating an entire back side of a substrate could lead to undue heating which could adversely affect sensitive biopolymers, given the intensity of illumination required. The present invention realizes that back lighting a transparent substrate during array fabrication can be accomplished by providing a mirror on the back side and illuminating and imaging from the front side on which droplets are deposited. The present invention further realizes that scanning illumination on the front side can avoid undue heating.
The present invention then, provides a method of fabricating a biopolymer array (for example, a polynucleotide or, more specifically, a DNA or RNA array). The method includes depositing droplets of fluid carrying the biopolymer or a biomonomer onto a front side of a transparent substrate. Light is directed through the substrate from the front side, back through a substrate back side and a first set of deposited droplets on the first side to an image sensor. In this manner, the first set is xe2x80x9cimagedxe2x80x9d. The light may optionally pass through the substrate from the front side at a position other than the first droplet set before being reflected to pass back through the back side of the substrate and first droplet set. Particularly, the light may pass through the substrate from the first side at an angle to a normal of the first side, and pass back through the back side and first droplet set at a complementary angle to the normal. Alternatively, the light may pass through the first droplet set when passing through the substrate from the first side, before being reflected to again pass through the first droplet set. In either event, the light is optionally reflected at a position spaced from the back side.
The directing of light in the foregoing manner may be repeated for additional sets of the deposited droplets by scanning the directed and reflected light across the first side. This can, for example, be accomplished by scanning both a light source of the directed light and the image sensor in unison across the first surface. Furthermore, the droplets may be deposited as droplet sets by a head, and multiple droplet sets may be deposited by scanning the head across the first side. Any deposited set may or may not be the same set that is later imaged by the sensor as a set. Further, the light source, image sensor and head are preferably physically interconnected and are scanned in unison across the first surface. The mirror preferably faces at least that area on the second side corresponding to that area on the first side across which droplet sets are deposited.
The substrate may, in one arrangement, be held in a chuck having multiple upstanding ribs to support the second side of the substrate. In this case, the mirror from which light is reflected may include multiple mirror segments extending along channels defined between the ribs. The head may be scanned across the first side of the substrate by scanning along the channels in turn. The direction of light from the source to the sensor may be on a plane oriented along (and preferably parallel to) the direction of the channels, and the source and sensor also scanned in a direction along (and preferably parallel to) the channels. This is particularly useful in the case where the light source and camera are positioned at so as to direct and receive the reflected light at complementary angles in the manner as described above. The head may be adjusted toward or away from the first side of the substrate independently of the sensor.
An apparatus which can execute a method of the present invention, is also provided. Such an apparatus includes a mount on which a transparent substrate can be mounted. A head is provided to deposit the fluid droplets onto the front side of the mounted substrate. A light source, reflector, and image sensor are also provided to execute the steps required by them, as described above. The apparatus may include other features as already described in connection with the method. For example, the apparatus may further include a transport system for the head, light source and image sensor, so as to move them in a manner as described, preferably including scanning in unison (with the head, light source, and image sensor being preferably physically interconnected as described above). A processor may also be provided to control the transport system as required and cause the head to dispense multiple droplet sets in co-ordination with relative movement of the head and substrate. Other elements may include the substrate mount in the form of a chuck, as described above.
Apparatus and methods of the present invention, can be also be used to deposit droplets of any other fluid moiety or moieties, and embodiments of the apparatus can be described by replacing xe2x80x9cbiopolymerxe2x80x9d, or similar terms with xe2x80x9cmoietyxe2x80x9d. Also, methods of the present invention can be executed without the deposition step in the event that the droplets were previously provided on the substrate.
The present invention can provide any one or more of a number of advantages. For example, the deposition of the droplets can be accurately observed against a transparent substrate. The imaging arrangement is relatively simple to construct and offers little interference with other components of a deposition apparatus, such as a chuck of the type described. The light source and camera scanning arrangement can offer rapid scanning with little interference from chuck components, such as the described ribs.